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Glycyrrhizin, silymarin, and ursodeoxycholic acid regulate a common hepatoprotective pathway in HepG2 cells.


Background: Glycyrrhizin, silymarin, and ursodeoxycholic acid are widely used hepatoprotectants for the treatment of liver disorders, such as hepatitis C virus infection, primary biliary cirrhosis, and hepatocellular carcinoma.

Purpose: The gene expression profiles of HepG2 cells responsive to glycyrrhizin, silymarin, and ursodeoxycholic acid were analyzed in this study.

Methods: HepG2 cells were treated with 25 pM hepatoprotectants for 24 h. Gene expression profiles of hepatoprotectants-treated cells were analyzed by oligonucleotide microarray in triplicates. Nuclear factor-[kappa]B (NF-[kappa]B) activities were assessed by luciferase assay.

Results: Among a total of 30,968 genes, 252 genes were commonly regulated by glycyrrhizin, silymarin, and ursodeoxycholic acid. These compounds affected the expression of genes relevant various biological pathways, such as neurotransmission, and glucose and lipid metabolism. Genes involved in hepatocarcinogenesis, apoptosis, and anti-oxidative pathways were differentially regulated by all compounds. Moreover, interaction networks showed that NF-[kappa]B might play a central role in the regulation of gene expression. Further analysis revealed that these hepatoprotectants inhibited NF-[kappa]B activities in a dose-dependent manner.

Conclusion: Our data suggested that glycyrrhizin, silymarin, and ursodeoxycholic acid regulated the expression of genes relevant to apoptosis and oxidative stress in HepG2 cells. Moreover, the regulation by these hepatoprotectants might be relevant to the suppression of NF-[kappa]B activities.




Ursodeoxycholic acid




Liver cancer is the second most common cause of cancer death worldwide, causing about 746,000 deaths in 2012. The prognosis of liver cancer is very poor and the estimated incidence of new cases is 782,000 in the less developed regions in 2012 (Ferlay et al. 2015). Several studies illustrate that constitutive nuclear factor-[kappa]B (NF-[kappa]B) activity plays a central role in the hepatic neoplastic progression through the upregulation of anti-apoptotic genes (Kucharczak et al., 2003). Moreover, the inhibition of NF-[kappa]B activation in hepatocytes retards and reduces the development of hepatocellular carcinoma in mice (DiDonato et al. 2012). Therefore, the inhibition of NF-[kappa]B activation might be an effective strategy to treat liver cancers.

Glycyrrhizin, the triterpenoid saponin from Glycyrrhiza glabra L. roots (licorice), consists of one molecule of glycyrrhetinic acid and two molecules of glucuronic acid. Glycyrrhizin exhibits various pharmacological effects, such as anti-inflammatory and protective effects in liver (Li et al. 2014). Therefore, glycyrrhizin analogs, such as magnesium isoglycyrrhizinate and stronger neo-minophagen C, are effective and safe for the treatment of patients with chronic liver disease and liver dysfunction (Mori et al. 1990; Mao et al. 2009).

Silymarin is a flavonolignan complex from Silybum marianum (L.) Gaertn. fruits. Silymarin comprises a number of flavonolignans, including silibinin (silybin A and silybin B). isosilybin A and B, silychristin A and B. silydianin, and other phenolic compounds (Wu et al. 2009). Silymarin exhibits anti-inflammatory and immunomodulatory effects and thus promotes the health of livers (Polyak et al. 2013). In addition, silymarin-type drugs like legalon have been used for the treatment of acute hepatitis and nonalcoholic fatty liver disease in patients (El-Kamary et al. 2009; Loguercio et al. 2012).

Ursodeoxycholic acid, a hydrophilic stereoisomer of chenodeoxycholic acid, is a major component of Chinese black bear's bile (Ohtsuki et al., 1992). Ursodeoxycholic acid is used to treat chronic cholestatic liver diseases, such as primary biliary cirrhosis and primary sclerosing cholangitis (Chapman 2009; Lindor et al. 2009). Moreover, some evidences indicate that ursodeoxycholic acid decreases the levels of alanine aminotransferase, aspartate aminotransferase, and gamma-glutamyl transpeptidase in patients with chronic hepatitis C and protects livers from against methotrexate-induced toxicity (Omata et al. 2007; Uraz et al. 2008).

Few reports have evaluated the genomic alterations elicited by glycyrrhizin, silymarin, and ursodeoxycholic acid. For examples, Clycyrrhiza glabra root extract induces the proliferation of MCF-7 cells by activating extracellular signal-regulated kinases 1/2 and Akt pathways (Dong et al., 2007). Treating hepatocytes with ursodeoxycholic acid shows that ursodeoxycholic acid affects the expression of genes directly involved in cell cycle and apoptotic events, and the E2F-1/p53/apoptotic protease activating factor-1 pathway seems to be the target of ursodeoxycholic acid (Castro et al. 2005). In this study, we treated hepatocytes with non-cytotoxic concentrations of glycyrrhizin, silymarin, and ursodeoxycholic acid, and analyzed the gene expression profiles by microarray. The gene expression profiles were further compared to evaluate the different and the common pathways regulated by these compounds.

Materials and methods

Cell culture

The human hepatoma cell line (HepG2) was obtained from Bioresource Collection and Research Center (Hsinchu, Taiwan). Recombinant HepG2/NF-[kappa]B cell, which carried the NF-[kappa]B-driven luciferase genes, was constructed as described previously (Hsiang et al. 2009). HepG2 cells and HepG2/NF-[kappa]B cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Gaithersburg, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT, USA), 100 [micro]g/ml streptomycin, and 100 unit/ml penicillin in a humidified incubator at 37[degrees]C with 5% C[O.sub.2].


Glycyrrhizin (purity [greater than or equal to] 95%), silymarin, and ursodeoxycholic acid (purity [greater than or equal to] 99%) were purchased from Sigma (St. Louis, MO, USA), dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 200 mM, and stored at -30[degrees]C. Silymarin (product number 254924) is a mixture of toxifolin (4%), silichristin (27.9%), silidianin (2.9%), silybin A (19.3%), silybin B (31.3%), isosilybin A (8.2%), and isosilybin B (2.3%). MG-132, a NF-[kappa]B inhibitor, was purchased from Santa Cruz (Dallas, TX, USA) and dissolved in DMSO to a final concentration of 50 mM. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma (St. Louis, MO, USA) and dissolved in phosphate-buffered saline (PBS) (137 mM NaCl, 1.4 mM KH2P04, 4.3 mM [Na.sub.2]HP[O.sub.4], 2.7 mM KCl, pH 7.2).

Total RNA isolation

HepG2 cells (2 x 106 cells, passage number 38) were seeded in a 25-[cm.sup.2] flask and incubated at 37[degrees]C for 24 h. Cells were then treated with 5 ml of culture medium containing 0.125%o DMSO (solvent control) or [micro]M hepatoprotectants, and incubated at 37[degrees]C for another 24 h. Total RNA was extracted from cells treated with or without compounds by RNeasy Mini kit (Qiagen, Valencia, CA, USA). Total RNA was quantified using the spectrophotometer (Beckman Coulter, Fullerton, CA, USA) and further evaluated using an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA sample with an RNA integrity number greater than 8.0 was accepted for microarray analysis.

Microarray analysis

Microarray analysis was performed as described previously (Lo et al. 2013; Ho et al. 2014). Briefly, fluorescent RNA targets were prepared from 5 [micro]g of total RNA samples using the MessageAmp[TM] aRNA kit (Ambion, Austin, TX, USA) and Cy5 dye (Amersham Pharmacia, Piscataway, NJ, USA). Fluorescent targets were hybridized to the Human Whole Genome OneArray[TM] (Phalanx Biotech Group, Hsinchu, Taiwan). Number of replicates was three. After an overnight hybridization at 50[degrees]C, non-specific targets were washed away and the array was scanned by an Axon 4000 scanner (Molecular Devices, Sunnyvale, CA, USA). Spots with a signal-to-noise ratio > 0 or control probes were selected and normalized by the R program of the limma package (Smyth and Speed 2003). We used surrogate variable analysis (sva) to capture the heterogeneity of expression caused by any variation and to improve the accuracy and reproducibility in analyzing gene expression levels (Leek and Storey 2007). Normalized data were tested by a standard paired t-test. The p-values were then adjusted for a false discovery rate (FDR) (Benjamini and Hochberg 1995). A value of FDR < 0.5 was considered statistically significant. The fold changes of genes were calculated by dividing the normalized signal intensities of genes in compound-treated cells by those in solvent-treated cells. Genes with fold changes [greater than or equal to] 2.0 or [less than or equal to] -2.0 and FDR values <0.5 were selected as differentially expressed genes and further analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway ( We used the Web-based gene set analysis toolkit ( to test the enriched pathways. Microarray data are minimum information about microarray experiments compliant, and raw data have been deposited in the Gene Expression Omnibus (accession number: GSE67504).

Quantitative real-time polymerase chain reaction (qPCR)

Total RNA was reverse-transcribed for 2 h at 37[degrees]C using High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). qPCR was performed by mixing cDNA, 2 x Power SYBR Green PCR Master kit and 200 nM of forward and reverse primers. The reaction condition was followed: 10 min at 95[degrees]C; 40 cycles of 15 s at 95[degrees]C and 1 min at 60[degrees]C. Each assay was run on an Applied Biosystems 7300 Real-Time PCR system in triplicates. Relative quantitation (RQ) was calculated using the comparative CT method ([DELTA][DELTA][C.sub.T]) which determines the change in expression of a nucleic acid sequence in a test sample (treated group) relative to the same sequence in a calibrator sample (mock group) (Livak and Schmittgen 2001). The [DELTA][C.sub.T] value is determined by subtracting the average [C.sub.T] value of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene from the average CT value of target gene. The [DELTA][DELTA][C.sub.T] value is determined by subtracting the ACT value of mock group from the [DELTA][C.sub.T] value of treated group. RQis calculated as 2~ [DELTA][DELTA][C.sub.T]. Fold changes were further presented as RQ if the RQ value was [greater than or equal to]1, or as -1/RQ if the RQ value is <1. The primer ser for each gene was followed: histone deacetylase 9 (HDAC9) forward, 5'-GCAAGAGGAGACAGAGACCG-3'; HDAC9 reverse, 5' - ACTTG G C ACTTCAC AAG G CT- 3'; minichromosome maintenance deficient 3 associated protein (MCM3AP) forward, 5'-CGCTTCCTCTGGTGGTTCTT-3'; MCM3AP reverse, 5'CTGCACTGCTTGCAAAACCT-3'; GAPDH forward, 5'-TCACCCACACTGTGCCCATCTATGA-3'; GAPDH reverse, 5'-GAGGAAGAGGATGCGGCAGTGG-3'.

Gene interaction network analysis

We constructed three interaction networks of genes with fold changes [greater than or equal to] 2.0 or [less than or equal to] -2.0 in each treatment group using Genomatix Applications software. Then, we merged genes and their interactions in either network into a union network and finally classified this network by GOlorize, a cytoscape plug-in

(Garcia et al. 2007). GOlorize first highlighted the nodes that belonged to the same class using color-coding and then constructed an enhanced visualization of the network using a class-directed layout algorithm. Finally, we used Cell Region-Based Rendering and Layout (Cerebral), a cytoscape plug-in, to generate a pathway-like representation of a network. Cerebral uses subcellular localization annotation to create a layered view of a cell, placing nodes in the region corresponding to the appropriate localization (Barsky et al. 2007).

Cell viability assay

HepG2 cells (5 x 106 cells) were cultivated in a 96-well culture plate. After a 24-h incubation at 37[degrees]C, various amounts of glycyrrhizin, silymarin, or ursodeoxycholic acid were added to confluent cell monolayer and incubated for another 24 h. MG-132 ([micro]M), a NF-[kappa]B inhibitor, was added to HepG2/NF-[kappa]B cells to evaluate the cytotoxicity. Cell viability was monitored by the MTT colorimetric assay as described previously (Cheng et al. 2009). Cell viability (%) was calculated by the equation: (OD of compound-treated cells/OD of solvent-treated cells) x 100.

Luciferase assay

HepG2/NF-[kappa]B cells (5 x 106 cells) were cultured in a 96-well culture plate at 37[degrees]C for 24 h, washed with DMEM, and treated with 25 [micro]M of MG-132 or various amounts of glycyrrhizin, silymarin, or ursodeoxycholic acid for another 24 h. Cells were then washed with ice-cold PBS, lysed with 30 ptl Triton lysis buffer (50 mM Tris-HCl, 1% Triton X-100, 1 mM dithiothreitol, pH 7.8), and collected with a cell scraper. Luciferase activity was measured as described previously (Cheng et al. 2009). Relative luciferase activity was calculated by dividing the relative luciferase unit (RLU) of compound-treated cells by the RLU of solvent-treated cells.

Statistical analysis

Data were presented as mean [+ or -] standard error. Data were analyzed by one-way ANOVA and post hoc Bonferroni test using SPSS Statistics version 20 (IBM, Armonk, NY, USA). A p value less than 0.05 was considered as statistically significant.


Microarray analysis of hepatoprotectant-regulated gene expression

In this study, we chose HepG2 cell, hepatocytes derived from hepatocellular carcinoma, as a cell model to study the gene expression profiles of hepatoprotectants. Cytotoxic studies were first performed. Fig. 1 shows that glycyrrhizin, silymarin, and ursodeoxycholic acid displayed no visible cytotoxic effects at a broad range of concentration (0.5-500 [micro]M). Previous studies indicated that silymarin at 25 [micro]M achieves the highest hepatoprotective effect in FL83B mouse liver cells and at a concentration exceeding 25 [micro]M abruptly increases cell damage in rat hepatocytes (Sainz-Pardo et al. 1994; Lo et al. 2014). In addition, up to 25 [micro]M of glycyrrhizin dose not induce cell death in human epithelial ovarian carcinoma cells (Lee et al. 2010). Therefore, we treated HepG2 cells with 25 [micro]M compounds to analyze novel or common biological pathways regulated by glycyrrhizin, silymarin, and ursodeoxycholic acid in this study.

Microarray data were analyzed by limma and sva packages to examine the differentially expressed genes in cells treated with various hepatoprotectants. We selected genes with fold changes [greater than or equal to] 2.0 or [less than or equal to] -2.0 and FDR values <0.5 as differentially expressed genes for further analysis. Among a total of 30,968 genes, glycyrrhizin, silymarin, and ursodeoxycholic acid downregulated the expression of 777, 684, and 797 genes, respectively. In addition, the expression of 497, 604, and 710 genes was upregulated by glycyrrhizin, silymarin, and ursodeoxycholic acid, respectively. A Venn diagram was used to classify genes those were specific or common in the comparisons. Fig. 1 shows that glycyrrhizin and silymarin affected 145 genes, silymarin and ursodeoxycholic acid affected 196 genes, and glycyrrhizin and ursodeoxycholic acid affected 227 genes. Furthermore, a total of 252 genes was commonly affected by glycyrrhizin, silymarin, and ursodeoxycholic acid.

KEGG pathway analysis of differentially expressed genes altered by three hepatoprotectants

We further analyzed the enriched pathways altered by three hepatoprotectants. Table 1 shows that various KEGG pathways were significantly regulated by glycyrrhizin, silymarin, and ursodeoxycholic acid. No common KEGG pathway was affected by these hepatoprotectants. Glycyrrhizin regulated calcium signaling and T cell receptor signaling pathways, silymarin affected the insulin signaling pathway, and ursodeoxycholic acid affected neuroactive ligand-receptor interaction, tight junction, long-term depression, and extracellular matrix-receptor interaction. Expression levels of genes with p values <0.05 in these pathways are displayed in Table 2.

Interaction network of hepatoprotectant-regulated genes

The relationship between differentially expressed genes affected by each compound was further analyzed by Genomatix Applications software. Genes were correlated based on a review of published data, the Genomatix Knowledge Base, and promoter DNA sequence analysis. Three interaction networks, corresponding to three hepatoprotectants-regulated genes, were constructed. Then, we merged three gene interaction networks using Cytoscape. As shown in Fig. 2, NF-[kappa]B was the node in the gene interaction network. A total of 127 genes was affected by all three compounds and associated with NF-[kappa]B in this network. The expression profiles of 127 NF-rB-connected genes were then clustered by hierarchical clustering analysis. Fig. 3 shows that genes upregulated by one compound had tendencies of upregulation by other compounds, and vice versa. Among 127 genes, 12 genes with fold changes [greater than or equal to] 2.0 or [less than or equal to] -2.0 were commonly regulated by glycyrrhizin, silymarin, and ursodeoxycholic acid. The gene expression levels of these genes are shown in Table 3. Furthermore, we used Cerebral to generate pathway-like representations of networks for glycyrrhizin, silymarin, and ursodeoxycholic acid treatments. As shown in Fig. 4, these hepatoprotectants affected NF-[kappa]B via regulating the same or distinct genes. For example, in the extracellular space, glycyrrhizin modulated NF-[kappa]B activity by regulating interleukin 4 (IL4), 1L23A, and fibroblast growth factor 10 (FGF10) genes (Fig. 4A), silymarin modulated NF-[kappa]B by regulating IL6, IL9, and collagen type 2 alpha 1 (COL2A1) genes (Fig. 4B), and ursodeoxycholic acid affected NF-[kappa]B by regulating IL4, 1LU, and FGF10 genes (Fig. 4C). In addition, all compounds affected NF-[kappa]B by regulating HDAC9, MCM3AP, Wilms' tumor 1 (WT1), and Spi-B transcription factor (SP1B) genes in the nucleus.

We further verified microarray data by qPCR. We quantified the expression levels of HDAC9 and MCM3AP genes because these genes are associated with hepatocarcinogenesis and are directly interacted with NF-[kappa]B (Wang et al. 2010; Ding et al. 2013). In comparison with mock, the expression of HDAC9 genes was downregulated by glycyrrhizin, silymarin, and ursodeoxycholic acid, with fold changes of -2.08, -11.11, and -3.23, respectively (Table 4). The expression levels of MCM3AP genes were also decreased by glycyrrhizin, silymarin, and ursodeoxycholic acid, with fold changes of -2.56, -1.75, and -2.17, respectively. These findings indicated that the expression of HDAC9 and MCM3AP genes was downregulated by these hepatoprotectants, which were consistent with the findings of microarray data.

Effects of hepatoprotectants on NF-[kappa]B activities in HepC2 cells

Because NF-[kappa]B may play a central role in the glycyrrhizin-, silymarin- and ursodeoxycholic acid-regulated gene network, we wondered whether these compounds were able to affect NF-[kappa]B activities. HepG2/NF-[kappa]B cells were then treated with various amounts of glycyrrhizin, silymarin, or ursodeoxycholic acid, and the luciferase activity was determined 24 h later. As shown in Fig. 5, MG-132, a well-known proteasome inhibitor that inhibits NF-[kappa]B activity by inhibiting ItcB degradation, suppressed significantly the NF-tcB activity in HepG2 cells. Glycyrrhizin, silymarin, and ursodeoxycholic acid suppressed NF-[kappa]B activities in a dose-dependent manner. The EC50 value of silymarin on the inhibition of NF-[kappa]B activity was 64 [+ or -] 9.17 [micro]M, while [EC.sub.50] values of glycyrrhizin and ursodeoxycholic acid were >500 [micro]M. No visible cytotoxic effects were observed. These findings indicate that glycyrrhizin, silymarin, and ursodeoxycholic acid inhibited NF-[kappa]B activities in HepG2 cells.


In this study, we investigated the gene expression profiles of HepG2 cells in response to glycyrrhizin, silymarin, and ursodeoxycholic acid treatments. Differentially expressed genes were categorized according to KEGG pathways, and we found that hepatoprotectant-regulated genes were associated with various biological pathways. For example, adrenoceptor alpha 1A, glutamate receptor ionotropic N-methyl D-aspartate 2A, and purinergic receptor P2X ligand-gated ion channel 4 in calcium signaling pathway, and glycine receptor alpha 1 and leptin receptor in neuroactive ligand-receptor interaction mediate actions in the nervous system through the binding of different neurotransmitters (Betz et al. 1999; Koshimizu et al. 2003; Vial et al. 2004; Ryan et al. 2008). Neuroblastoma RAS viral oncogene homolog and phosphoinositide-3-kinase regulatory subunit 1 in T cell receptor signaling pathway are also involved in insulin signaling pathway (Saltiel and Kahn 2001). Protein kinase AMP-activated beta 1 (PRKAB1) and PRKAG3 are known to be associated with lipogenesis (Bailey 2007). Based on these results, we suggested that these hepatoprotectants might regulate pathways involved in neurotransmission, and glucose and lipid metabolism in liver.

We further investigated differentially expressed genes by gene interaction network analysis. We found that NF-[kappa]B seemed to be in the central part of the network and 127 genes were connected directly to NF-[kappa]B. Furthermore, we found that 12 NF-[kappa]B-connected genes were differentially expressed in all three compound treatments. Among them, HDAC9, MCM3AP, SPIB, WT1, ethylmalonic encephalopathy 1 (ETHE1), BH3 interacting domain death agonist (BID), superoxide dismutase 2 (SOD2), and mitogen-activated protein kinase kinase kinase 7 (MAP3K7) genes are known to be involved in hepatocarcinogenesis, apoptosis, and anti-oxidative pathways, and their expressions are directly regulated by NF-[kappa]B. For examples, HDAC9 is a transcriptional regulator of the histone deacetylase family, which is regulated by Sol and NF-[kappa]B (Ma et al. 2011). Recent study indicated that HDAC9 promotes hepatocellular carcinoma progression by inhibiting p53 transcriptional activity (Ding et al. 2013). MCM3AP is an acetyltransferase that acetylases MCM3, and plays a role in DNA replication (Takei et al. 2001). It is also a novel hepatitis B virus integration site, and upregulation of MCM3AP promotes the hepatocarcinogenesis by affecting flanking sequences (Wang et al. 2010). SPIB is a transcription factor that binds to a purine-rich sequence of promoters (Bonadies et al. 2010). Using a random-walk-based community detection algorithm, Petrochilos et al. (2013) have identified that SPIB is functionally related to cancer and shows a promise as a therapeutic target. WT1 encodes a transcription factor involved in cell growth and development. The presence of NF-[kappa]B-responsive elements in the promoter of WT1 gene suggests that the transcription of WT1 gene is regulated by NF-[kappa]B pathway (Dehbi et al. 1998). WT1 is expressed in a substantial proportion of hepatocellular carcinoma contributing to tumor progression and resistance to chemotherapy, suggesting that WT1 may be an important target for liver cancer treatment (Perugorria et al. 2009). ETHE1 gene accelerates the export of NF-[kappa]B from nucleus and inhibits p53-dependent apoptosis, thus contributing to hepatocarcinogenesis (Higashitsuji et al. 2002). In our study, all three compounds reduced the expressions of HDAC9, MCM3AP, SPIB, WT1, and ETHE1 genes at non-toxic concentrations in hepatocytes, suggesting the anticancer potentials of these compounds.

In addition to ETHE1, BID gene product is a specific proximal substrate of caspase-8 in the Fas apoptotic signaling pathway (Li et al. 1998). While full-length BID is localized in cytosol, truncated BID (tBID) translocates to mitochondria and thus transduces apoptotic signals from cytoplasmic membrane to mitochondria. The SOD2 gene encodes an intramitochondrial free radical scavenging enzyme, the first line of defense against superoxide produced by oxidative phosphorylation. SOD2 maintains the integrity of mitochondrial enzymes that are inactivated by superoxide (Li et al. 1995). SOD2 and tBID are known to be associated with apoptosis via a mitochondrial pathway (Yin 2000; Pardo et al. 2006). The ability of glycyrrhizin, silymarin, and ursodeoxycholic acid to reduce BID or induce SOD2 may protect HepG2 cells from damage by suppressing apoptosis via the mitochondrial pathway.

MAP3K7 is linked to IL1 receptor and tumor necrosis factor (TNF) receptor signalings (Sato et al. 2005). MAP3K7 is activated by chemical and physical stresses and regulates stress-induced activation of activator protein-1 and NF-[kappa]B (Ishitani et al. 2003; Takaesu et al. 2003; Shim et al. 2005). Hypoxia-activated MAP3K7 results in the activation of c-Jun-dependent transcription in response to oxidative stress (Blanco et al. 2007). In addition, BID is also able to induce the generation of reactive oxygen species (ROS) following death receptor activation (Ding et al. 2004). However, SOD2 plays a critical cytoprotective role against oxidative stress (Delhalle et al. 2002). In this study, the up-regulation of SOD2 and the down-regulation of BID and TAK1 by glycyrrhizin, silymarin, and ursodeoxycholic acid suggested the anti-oxidative properties of these compounds.

Hepatocytes exposed to BID siRNA are resistant to Fas and TNF-[alpha]-induced cell death (Yin et al. 1999; Guicciardi et al. 2005). Treatment of lymph-proliferative mice with BID antisense also ameliorates liver injury following bile duct ligation (Higuchi et al. 2001). Additionally, overexpression of SOD2 is effective against mitochondrial oxidative stress in hepatocytes and is protective against alcohol-induced liver injury in an enteral feeding rat model (Wheeler et al. 2001). The up-regulation of SOD2 in hepatocytes is associated with a reduction of ROS-induced injury after ischemia-reperfusion (Chen et al. 2006). Dominant-negative MAP3K7 induces GO exit in the quiescent liver and accelerates hepatic cell cycle progression during regeneration (Bradham et al. 2001). Moreover, the inhibition of MAP3K7/c-Jun N-terminal kinase decreases the proliferation of hepatic stellate cells, a critical effector in hepatic fibrogenesis (Schnabi et al. 2001). Therefore, the differential expression of BID, SOD2, and TAK1 genes by glycyrrhizin, silymarin, and ursodeoxycholic acid suggested the hepatoprotective effects in livers.

NF-[kappa]B signaling pathways have a central function in the regulation of inflammation-fibrosis-cancer axis. NF-[kappa]B activation is crucially involved both in the fibrogenesis and in the initiation and promotion of liver cancers (Sun and Karin 2008; Luedde and Schwabe 2011). However, the dual role of NF-[kappa]B activation in hepatocarcinogenesis indicates that activation of NF-[kappa]B not only displays beneficial effects but also suppresses the viability of hepatocytes. For examples, in the early stages of tumorigenesis, NF-[kappa]B activation prevents the death of hepatocytes and thus avoids the release of proinflammatory cytokines by necrotic hepatocytes. However, in the late stages of tumorigenesis, NF-[kappa]B activation promotes the survival of transformed hepatocytes and elicits the malignancy of liver cancers (Sun and Karin 2008; Luedde and Schwabe 2011). Glycyrrhizin, silymarin, and ursodeoxycholic acid inhibited NF-[kappa]B activities in HepG2 cells, a human hepatoblastoma cell line, suggesting that glycyrrhizin, silymarin, and ursodeoxycholic acid might exhibit hepatoprotective effects by altering the survival of hepatoblastoma cells. Moreover, this finding was also consistent with previous reports that indicate glycyrrhizin, silymarin, and ursodeoxycholic acid play important roles in the treatment of hepatocellular carcinoma by reducing tumor cell proliferation or reducing the angiogenesis of liver (Kuiper et al. 2010; Li et al. 2014; Mastron et al. 2015).

In conclusions, this study demonstrated that different hepatoprotectants regulated different biological pathways. However, these hepatoprotectants also regulated the expression of common genes through a central molecule, NF-[kappa]B. Moreover, non-toxic dosages of hepatoprotectants inhibited NF-[kappa]B activities. Because the genes regulated by hepatoprotectants are relevant to antioxidant or antiapoptotic pathways, we proposed that these compounds exerted their hepatoprotective effects by regulating apoptosis and oxidative stress in hepatocarcinoma cells.

Conflict of interest

There was no conflict of interest.

Abbreviations: NF-[kappa]B, nuclear factor-[kappa]B; DMEM, Dulbecco's modified Eagle's medium: DMSO, dimethyl sulfoxide: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; sva, surrogate variable analysis; FDR, false discovery rate; KEGG, Kyoto Encyclopedia of Genes and Genomes; qPCR, quantitative real-time polymerase chain reaction; RQ, relative quantitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDAC9, histone deacetylase 9; MCM3AP, minichromosome maintenance deficient 3 associated protein; Cerebral, cell region-based rendering and layout; RLU, relative luciferase unit; IL, interleukin; FGF10, fibroblast growth factor 10; COL2A1, collagen type 2 alpha 1; SPIB, Spi-B transcription factor; PRKAB1, protein kinase AMP-activated beta 1; ETHE1, ethylmalonic encephalopathy 1; BID, BH3 interacting domain death agonist; SOD2, superoxide dismutase 2; MAP3K7, mitogen-activated protein kinase kinase kinase 7; tBID, truncated BID; TNF, tumor necrosis factor; ROS, reactive oxygen species.


Article history:

Received 29 August 2014

Revised 18 May 2015

Accepted 19 May 2015


This work was supported by grants from Ministry of Science and Technology (NSC101-2320-B-039-034-MY3 NSC102-2632-B-039-001-MY3), China Medical University (CMU102-NSC-04 and CMU103-SR-44), and CMU under the Aim for Top University Plan of the Ministry of Education, Taiwan.


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Chien-Yun Hsiang (a,1), Li-Jen Lin (b,1), Shung-Te Kao (b), Hsin-Yi Lo (c), Shun-Ting Chou (c), Tin-Yun Ho (c,d) *

(a) Department of Microbiology, China Medical University, Taichung 40402, Taiwan

(b) School of Chinese Medicine, China Medical University, Taichung 40402, Taiwan

(c) Graduate Institute of Chinese Medicine, China Medical University, Taichung 40402, Taiwan

(d) Department of Health and Nutrition Biotechnology, Asia University, Taichung 41354, Taiwan

* Corresponding author at: Graduate Institute of Chinese Medicine, China Medical University, 91 Hsueh-Shih Road, Taichung 40402, Taiwan.

Tel.: +886 4 2205 3366x3302; fax: +886 4 2203 2295.

E-mail address: (T.-Y. Ho).

(1) These authors contributed equally to this work.

Table 1
Classification of genes altered by glycyrrhizin, silymarin, or
ursodeoxycholic acid in HepG2 cells by KEGG pathways.

Compound               Pathway                      Observed   p-value

Glycyrrhizin           Calcium signaling pathway    13 (176)   0.00098
                       T cell receptor signaling     8 (93)    0.00416
Silymarin              insulin signaling pathway    10 (135)   0.00392
Ursodeoxycholic acid   Neuroactive                  22 (254)   0.00037
                       Tight junction               10 (136)   0.00496
                       Long-term depression          8 (76)    0.00429
                       ECM-receptor interaction      8 (87)    0.00679

Table 2
Expression levels of genes which belong to glycyrrhizin-,
silymarin-, or ursodeoxycholic acid-altered KEGG pathways in HepG2

Compound          Gene name           Gene description

Glycyrrhizin      Calcium signaling
                  ADRA1A              Adrenoceptor alpha 1A
                  SLC25A4             Solute carrier family 25,
                                        member 4
                  CACNA1E             Calcium channel,
                                        voltage-dependent, alpha IE
                  GRIN2A              Glutamate receptor, ionotropic,
                                        N-methyl D-aspartate 2A
                  P2RX4               Purinergic receptor P2X,
                                        ligand-gated ion channel 4
                  PPID                Peptidylprolyl isomerase D
                  T cell receptor
                  CD3Z                CD3Z antigen, zeta polypeptide
                  IL4                 Interleukin 4
                  NRAS                Neuroblastoma RAS viral (v-ras)
                                        oncogene homolog
                  PIK3R1              Phosphoinositide-3-kinase,
                                        regulatory subunit 1
Silymarin         Insulin signaling
                  PDE3A               Phosphodiesterase 3A,
                  PRKAB1              Protein kinase, AMP-activated,
                                        beta 1 non-catalytic subunit
                  RPS6                Ribosomal protein S6
                  PRKAG3              Protein kinase, AMP-activated,
                                        gamma 3 non-catalytic subunit
Ursodeoxycholic   Neuroactive
  acid              ligand-receptor
                  GLRA1               Glycine receptor, alpha 1
                  LEPR                Leptin receptor
                  PPP2CB              Protein phosphatase 2,
                                        catalytic subunit, beta

Compound          Gene name           Change (folds)        p-value

Glycyrrhizin      Calcium signaling
                  ADRA1A              -4.00 [+ or -] 1.36   0.0264
                  SLC25A4             -2.23 [+ or -] 0.54   0.0456
                  CACNA1E              2.87 [+ or -] 0.72   0.0248
                  GRIN2A               2.64 [+ or -] 0.44   0.0101
                  P2RX4               -3.47 [+ or -] 1.25   0.0410
                  PPID                -2.22 [+ or -] 0.34   0.0144
                  T cell receptor
                  CD3Z                -2.17 [+ or -] 0.48   0.0384
                  IL4                  2.10 [+ or -] 0.36   0.0229
                  NRAS                -2.43 [+ or -] 0.20   0.0017
                  PIK3R1               -236 [+ or -] 0.40   0.0151
Silymarin         Insulin signaling
                  PDE3A               -2.45 [+ or -] 0.34   0.0076
                  PRKAB1              -4.91 [+ or -] 0.56   0.0008
                  RPS6                 2.33 [+ or -] 0.45   0.0225
                  PRKAG3              -2.22 [+ or -] 0.32   0.0112
Ursodeoxycholic   Neuroactive
  acid              ligand-receptor
                  GLRA1                3.72 [+ or -] 0.75   0.0230
                  LEPR                 2.19 [+ or -] 0.30   0.0284
                  PPP2CB              -2.75 [+ or -] 0.34   0.0147

Table 3
Expression levels of 12 common NF-[kappa]B-connected genes in HepG2
cells responsive to glycyrrhizin, silymarin, and ursodeoxycholic acid

Gene name   Gene description

MAP3K7      Mitogen-activated protein kinase kinase kinase 7
HDAC9       Histone deacetylase 9
CFTR        Cystic fibrosis transmembrane conductance regulator
ETHE1       Ethylmalonic encephalopathy 1
MCM3AP      Minichromosome maintenance deficient 3 associated protein
SPIB        Spi-B transcription factor
WT1         Wilms' tumor 1
BID         BH3 interacting domain death agonist
C1QTNF3     Clq and tumor necrosis factor related protein 3
S0D2        Superoxide dismutase 2
SFRS1       Splicing factor, arginine/serine-rich 1
UBE2N       Ubiquitin-conjugating enzyme E2N

Gene name   Glycyrrhizin                 Silymarin

            Change (folds)        FDR    Change (folds)        FDR

MAP3K7      -5.98 [+ or -] 3.61   0.06   -5.71 [+ or -] 3.21   0.05
HDAC9       -3.93 [+ or -] 1.24   0.02   -4.92 [+ or -] 3.68   0.12
CFTR        -3.64 [+ or -] 1.06   0.02   -2.43 [+ or -] 0.91   0.10
ETHE1       -3.07 [+ or -] 0.83   0.03   -2.73 [+ or -] 0.87   0.05
MCM3AP      -2.92 [+ or -] 1.56   0.14   -2.17 [+ or -] 1.02   0.20
SPIB        -2.78 [+ or -] 0.88   0.05   -3.34 [+ or -] 2.65   0.23
WT1         -2.43 [+ or -] 1.39   0.22   -2.09 [+ or -] 1.44   0.36
BID         -2.20 [+ or -] 1.48   0.33   -2.58 [+ or -] 2.52   0.40
C1QTNF3      2.02 [+ or -] 0.75   0.15    2.74 [+ or -] 2.29   0.31
S0D2         2.43 [+ or -] 1.27   0.19    3.01 [+ or -] 2.22   0.23
SFRS1        2.97 [+ or -] 2.36   0.26    2.38 [+ or -] 2.59   0.48
UBE2N        4.24 [+ or -] 1.79   0.04    2.55 [+ or -] 1.23   0.15

Gene name   Ursodeoxycholic acid

            Change (folds) FDR

MAP3K7      -4.59 [+ or -] 1.83   0.06
HDAC9       -3.81 [+ or -] 0.65   0.02
CFTR        -7.41 [+ or -] 7.75   0.20
ETHE1       -3.11 [+ or -] 1.47   0.14
MCM3AP      -2.74 [+ or -] 1.85   0.27
SPIB        -2.33 [+ or -] 0.58   0.08
WT1         -2.10 [+ or -] 1.01   0.26
BID         -7.37 [+ or -] 5.26   0.11
C1QTNF3      2.76 [+ or -] 1.86   0.27
S0D2         3.02 [+ or -] 2.46   0.31
SFRS1        3.50 [+ or -] 2.68   0.24
UBE2N        4.71 [+ or -] 0.98   0.02

Table 4
Expression levels of HDAC9 and MCM3AP genes in glycyrrhizin-,
silymarin-, and ursodeoxycholic acid-treated HepG2 cells by qPCR.

Gene     Sample            Avg [C.sub.T]         Average [C.sub.T]
                                                      of GAPDH

HDAC9    Glycyrrhizin      26.47 [+ or -] 0.06   30.04 [+ or -] 0.01
         Silymarin         37.07 [+ or -] 1.07   30.13 [+ or -] 0.03
         Ursodeoxycholic   27.37 [+ or -] 0.13   30.13 [+ or -] 0.03
MCM3AP   Glycyrrhizin      33.89 [+ or -] 1.06   30.04 [+ or -] 0.01
         Silymarin         33.52 [+ or -] 1.19   30.07 [+ or -] 0.01
         Ursodeoxycholic   33.90 [+ or -] 1.06   30.13 [+ or -] 0.03

Gene     Sample            [DELTA][C..sub.T] (a)

HDAC9    Glycyrrhizin      -3.41 [+ or -] 0.17
         Silymarin          6.94 [+ or -] 1.07
         Ursodeoxycholic   -2.76 [+ or -] 0.13
MCM3AP   Glycyrrhizin       4.00 [+ or -] 1.07
         Silymarin          3.44 [+ or -] 1.19
         Ursodeoxycholic    3.77 [+ or -] 1.06

Gene     Sample            [DELTA] [DELTA][C.sub.T] (b)

HDAC9    Glycyrrhizin      1.05 [+ or -] 0.17
         Silymarin         3.48 [+ or -] 1.07
         Ursodeoxycholic   1.70 [+ or -] 0.13
MCM3AP   Glycyrrhizin      1.36 [+ or -] 1.07
         Silymarin         0.81 [+ or -] 1.19
         Ursodeoxycholic   1.13 [+ or -] 1.06

Gene     Sample            Change (folds) (c)

HDAC9    Glycyrrhizin       -2.08
         Silymarin         -11.11
         Ursodeoxycholic    -3.23
MCM3AP   Glycyrrhizin       -2.56
         Silymarin          -1.75
         Ursodeoxycholic    -2.17

(a) The [DELTA][C.sub.T] value is determined by subtracting the
average [C.sub.T] value of GAPDH gene from the average [C.sub.T]
value of target gene. The standard deviation of the difference is
calculated from the standard deviations of the target gene and GAPDH

(b) The [DELTA] [DELTA][C.sub.T] value is determined by subtracting
the [DELTA][C.sub.T] value of mock group from the [DELTA][C.sub.T]
value of treated group. This is a subtraction of an arbitrary
constant, so the standard deviation of [DELTA] [DELTA][C.sub.T] is
the same as the standard deviation of [DELTA][C.sub.T] value.

(c) RQ is calculated as [2.sup.[DELTA][DELTA]CT]. Fold changes were
presented as RQif the RQ value was [greater than or equal to] 1, and
as -1/RQif the RQ value is <1.
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Author:Hsiang, Chien-Yun; Lin, Li-Jen; Kao, Shung-Te; Lo, Hsin-Yi; Chou, Shun-Ting; Ho, Tin-Yun
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Jul 15, 2015
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